In eukaryotes, stress rapidly triggers the conjugation of small ubiquitin-related modifier (SUMO) to intracellular target proteins to alter their activity and mount a defense response. Defects in stress-induced SUMOylation (SIS) are associated with a number of human diseases including acute and chronic liver disease and cancer induced by genotoxic stress. In addition, several pathogenic bacteria (Listeria monocytogenes, Streptococcus pneumoniae) and viruses (human adenovirus, influenza virus) alter SIS, and this is important for bypassing defenses and promoting infection. Like metazoans, several plant pathogens hijack the SUMOylation pathway to render host defense ineffectual, suggesting that this pathway serves a conserved role in defense against biotic attack. Thus, it is essential that we determine precisely how SUMOylation triggers changes that minimize stress damage, inhibit pathogenesis, and promote survival. In particular, a long-term objective of this study is to determine how the SIS response mediates downstream effects that lead to stress protection. The flowering plant Arabidopsis thaliana is well suited for understanding the role of SUMOylation during stress. It has characterized mutants affecting the SIZ1 ligase that drives SIS, which are hypersensitive to many stresses and fail to elicit the SIS response; thereby permitting the dissection of SUMOylation during stress. In addition, substantial overlap between SUMO targets in plants and metazoans suggests a conserved response that converges on factors important for transcriptional regulation, RNA metabolism, and chromatin modification, indicating that the SIS response universally affects gene expression and chromatin accessibility. To identify the transcriptional and chromatin alterations associated with the SIS response, comparisons of wild type and siz1 mutant plants will be performed before and after heat stress. Specifically, next-generation sequencing will be used to track changes in SUMO1/2 binding occupancy along chromatin (ChIP-Seq) and the alterations to mRNA abundance/splicing (RNA-Seq). Changes occurring in wild type plants that are absent in siz1 mutants will define how SUMOylation rewires the genome in response to stress and, in doing so, has the potential to identify novel genes involved in stress tolerance. Some SUMO targets are also modified with ubiquitin specifically during stress with their dynamics suggesting that they are targeted for degradation. A second focus of this proposal aims to identify dual- modified targets using a novel proteomics approach. These targets will assist in identifying candidate SUMO- targeted ubiquitin ligases that catalyze this reaction. Loss-of-function analyses before and after heat stress will establish the importance and function of this dual modification during adverse conditions. Collectively, these experiments aim to provide a comprehensive understanding of the outputs and regulation of the SIS response. Broadly, this work will provide a mechanistic basis for understanding how organisms can rapidly respond and defend themselves from environmental stress and pathogen invasion.